Pocai et al (Diabetes 2009, 58, 2258-2266), Day et al. (Nat Chem Biol 2009, 5, 749-757) and Kosinski et al. (Obesity 2012, 20, 1566-1571) describe dual agonists of the glucagon-like peptide-1 (GLP-1) and glucagon receptors, e.g. by combining the actions of GLP-1 and glucagon in one molecule, which lead to a therapeutic principle with anti-diabetic action and a pronounced weight lowering effect superior to pure GLP-1 agonists, among others due to glucagon-receptor mediated increased satiety and energy expenditure.
Hoist (Physiol, Rev. 2007, 87, 14091439) and Meier (Nat. Rev. Endocrinol. 2012, 8, 728-742) describe that GLP-1 receptor agonists, such as GLP-1, liraglutide and exendin-4, have 3 major pharmacological activities to improve glycemic control in patients with T2DM (type 2 diabetes mellitus) by reducing fasting and postprandial glucose (FPG and PPG): (i) increased glucose-dependent insulin secretion (improved first- and second-phase), (ii) glucagon suppressing activity under hyperglycemic conditions, and (iii) delay of gastric emptying rate resulting in retarded absorption of meal-derived glucose.
The amino acid sequence of GLP-1(7-36)-amide is shown as SEQ ID NO: 1.
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
Liraglutide is a marketed chemically modified GLP-1 analog in which, among other modifications, a fatty acid is linked to a lysine in position 20 leading to a prolonged duration of action (Drucker et al, Nat. Rev. Drug Disc. 2010, 9, 267-268; Buse et al., Lancet 2009, 374, 39-47).
The amino acid sequence of Liraglutide is shown as SEQ ID NO: 2.
HAEGTFTSDVSSYLEGQAAK((S)-4-Carboxy-4- hexadecanoylamino-butyryl-)EFIAWLVRGRG-OH
Glucagon is a 29-amino acid peptide which is released into the bloodstream when circulating glucose is low. Glucagon's amino acid sequence is shown as SEQ ID NO: 3.
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH
During hypoglycemia, when blood glucose levels drop below normal, glucagon signals the liver to break down glycogen and release glucose, causing an increase of blood glucose levels to reach a normal level. Recent publications suggest that glucagon has in addition beneficial effects on reduction of body fat mass, reduction of food intake, and increase of energy expenditure (see e.g. Heppner et al., Physiology & Behavior 2010, 100, 545-548).
GIP (glucose-dependent insulinotropic polypeptide) is a 42 amino acid peptide that is released from intestinal K-cells following food intake. GIP and GLP-1 are the two gut enteroendocrine cell-derived hormones accounting for the incretin effect, which accounts for over 70% of the insulin response to an oral glucose challenge (Baggio et al. Gastroenterology 2007, 132, 2131-2157).
GIP's amino acid sequence is shown as SEQ ID NO: 5.
YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-OH
Peptides which are based on the structures of GLP-1 or glucagon, and bind and activate both the glucagon and the GLP-1 receptor (Hjorth et al. Journal of Biological Chemistry, 1994, 269, 30121-30124; Day et al, Nature Chem Biol, 2009, 5, 749-757) and suppress body weight gain and reduce food intake are described in patent applications WO 2008/071972, WO 2008/101017, WO 2009/155258, WO 2010/096052, WO 2010/096142, WO 2011/075393, WO 2008/152403, WO 2010/070251, WO 2010/070252, WO 2010/070253, WO 2010/070255, WO 2011/160630, WO 2011/006497, WO 2011/087671, WO 2011/087672, WO 2011/117415, WO 2011/117416, WO 2012/150503, WO 2012/158965, WO 2012/177444, WO 2013/004983, WO 2013/092703, WO 2014/017843, WO 2014/041195, WO 2014/041375, WO 2014/081872, WO 2014/140222, WO 2014/170496, WO 2015/055801, WO 2015/055802, WO 2015/132599, WO 2016/045400, WO 2016/055610 and WO 2016/065090, the contents of which are herein incorporated by reference. Other peptidic dual agonists are described in WO 2012/177443, WO 2013/186240, WO 2014/056872, WO 2014/091316, WO 2015/086731, WO 2015/086732 and WO 2015/086733, the contents of which are herein incorporated by reference. The body weight reduction was shown to be superior to pure GLP-1 agonists.
Exendin-4 is a 39 amino acid peptide which is produced by the salivary glands of the Gila monster (Heloderma suspectum) (Eng et al., J. Biol. Chem. 1992, 267, 7402-7405). Exendin-4 is an activator of the GLP-1 receptor, whereas it shows low activation of the GIP receptor and does not activate the glucagon receptor (see Table 1).
TABLE 1Potencies of exendin-4 at human GLP-1, GIP and Glucagon receptors(indicated in pM) at increasing concentrations and measuring theformed cAMP as described in Methods.SEQIDEC50 hGLP-1 REC50 hGIP REC50 hGlucagonNO:peptide[pM][pM]R [pM]4exendin-40.412500.0>10000000
The amino acid sequence of exendin-4 is shown as SEQ ID NO: 4.
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2
Exendin-4 shares many of the glucoregulatory actions observed with GLP-1. Clinical and nonclinical studies have shown that exendin-4 has several beneficial antidiabetic properties including a glucose dependent enhancement in insulin synthesis and secretion, glucose dependent suppression of glucagon secretion, slowing down gastric emptying, reduction of food intake and body weight, and an increase in beta-cell mass and markers of beta cell function (Gentilella et al., Diabetes Obes Metab., 2009, 11, 544-556; Norris et al, Diabet Med., 2009, 26, 837-846; Bunck et al, Diabetes Care., 2011, 34, 2041-2047).
These effects are beneficial not only for diabetics but also for patients suffering from obesity. Patients with obesity have a higher risk of getting diabetes, hypertension, hyperlipidemia, cardiovascular and musculoskeletal diseases.
Relative to GLP-1, exendin-4 is resistant to cleavage by dipeptidyl peptidase-4 (DPP4) resulting in a longer half-life and duration of action in vivo (Eng J., Diabetes, 1996, 45 (Suppl 2):152A (abstract 554)).
Exendin-4 was also shown to be much more stable towards degradation by neutral endopeptidase (NEP), when compared to GLP-1, glucagon or oxyntomodulin (Druce et al., Endocrinology, 2009, 150(4), 1712-1722). Nevertheless, exendin-4 is chemically labile due to methionine oxidation in position 14 (Hargrove et al., Regul. Pept., 2007, 141, 113-119) as well as deamidation and isomerization of asparagine in position 28 (WO 2004/035623).
Bloom et al. (WO 2006/134340) disclose that peptides which bind and activate both the glucagon and the GLP-1 receptor can be constructed as hybrid molecules from glucagon and exendin-4, where the N-terminal part (e.g. residues 1-14 or 1-24) originates from glucagon and the C-terminal part (e.g. residues 15-39 or 25-39) originates from exendin-4. Such peptides comprise glucagon's amino acid motif YSKY in positions 10-13. Krstenansky et al (Biochemistry, 1986, 25, 3833-3839) show the importance of these residues 10-13 of glucagon for its receptor interactions and activation of adenylate cyclase.
In the exendin-4 derivatives described in this invention, several of the underlying residues are different from glucagon and the peptides described in WO 2006/134340. In particular residues Tyr10 and Tyr13, which are known to contribute to the fibrillation of glucagon (J S Pedersen et al., Biochemistry, 2006, 45, 14503-14512) are replaced by Leu in position 10 and Gln, a non-aromatic polar amino acid, in position 13. This replacement, especially in combination with isoleucine in position 23 and glutamate in position 24, leads to exendin-4 derivatives with potentially improved biophysical properties as solubility or aggregation behaviour in solution. The non-conservative replacement of an aromatic amino acid with a polar amino acid in position 13 of an exendin-4 analogue leads to peptides with high activity on the glucagon receptor, keeping their activity on the GLP-1 receptor (see also WO 2013/186240 and WO 2014/056872).